The Aromatic "Trapping" of the Catalytic Histidine Is Essential for Efficient Catalysis in Acetylcholinesterase
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- 作者：Dov Barak ; Dana Kaplan ; Arie Ordentlich ; Naomi Ariel ; Baruch Velan ; and Avigdor Shafferman
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：July 2, 2002
- 年：2002
- 卷：41
- 期：26
- 页码：8245 - 8252
- 全文大小：234K
- 年卷期：v.41,no.26(July 2, 2002)
- ISSN：1520-4995
文摘
While substitution of the aromatic residues (Phe295, Phe338), located in the vicinity of thecatalytic His447 in human acetylcholinesterase (HuAChE) had little effect on catalytic activity, simultaneousreplacement of both residues by aliphatic amino acids resulted in a 680-fold decrease in catalytic activity.Molecular simulations suggested that the activity decline is related to conformational destabilization ofHis447, similar to that observed for the hexamutant HuAChE which mimics the active center ofbutyrylcholinesterase. On the basis of model structures of other cholinesterases (ChEs), we predicted thatcatalytically nonproductive mobility of His447 could be restricted by introduction of aromatic residue ina different location adjacent to this histidine (Val407). Indeed, the F295A/F338A/V407F enzyme is 170-fold more reactive than the corresponding double mutant and only 3-fold less reactive than the wild-typeHuAChE. However, analogous substitution of Val407 in the hexamutant HuAChE (generating theheptamutant Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F) did not enhance catalytic activity.Reactivity of these double, triple, hexa, and hepta mutant HuAChEs was monitored toward covalent ligandssuch as organophosphates and the transition state analogue TMFTA, which probe, respectively, the facilityof the enzymes to accommodate Michaelis complexes and to undergo the acylation process. The findingssuggest that in the F295A/F338A mutant the two His447 conformational states, which are essential forthe different stages of the catalytic process, seem to be destabilized. On the other hand, in the F295A/F338A/V407F mutant only the state involved in acylation is impaired. Such differential effects on theHis447 conformational properties demonstrate the general role of aromatic residues in cholinesterases,and probably in other serine hydrolases, in "trapping" of the catalytic histidine and thereby in optimizationof catalytic activity.